Microfluidic particle dam for direct visualization of SARS-CoV-2 antibody levels in COVID-19 vaccinees

Various COVID-19 vaccines are currently deployed, but their immunization varies and decays with time. Antibody level is a potent correlate to immune protection, but its quantitation relies on intensive laboratory techniques. Here, we report a decentralized, instrument-free microfluidic device that directly visualizes SARS-CoV-2 antibody levels. Magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) can bind to SARS-CoV-2 antibodies simultaneously. In a microfluidic chip, this binding reduces the incidence of free PMPs escaping from magnetic separation and shortens PMP accumulation length at a particle dam. This visual quantitative result enables use in either sensitive mode [limit of detection (LOD): 13.3 ng/ml; sample-to-answer time: 70 min] or rapid mode (LOD: 57.8 ng/ml; sample-to-answer time: 20 min) and closely agrees with the gold standard enzyme-linked immunosorbent assay. Trials on 91 vaccinees revealed higher antibody levels in mRNA vaccinees than in inactivated vaccinees and their decay in 45 days, demonstrating the need for point-of-care devices to monitor immune protection.

. Flow cytometry for optimization of protein immobilization of microparticles. The fluorescence intensity of (A) MMPs with a varied amount of anti-human IgG antibody and (B) PMPs with a varied amount of spike protein (mean ± SD, n = 10,000).

Fig. S2. The layout of the microfluidic channels.
After passing the magnetic separator, the PMPs (diameter of 15.34 μm) are trapped at the particle dam with the narrowest nozzle width of 8 μm. Because the channel height is 25 ± 0.3 μm, PMPs would accumulate as a monolayer in the trapping channel with a length inversely proportional to the amount of anti-spike IgG or anti-spike RBD IgG and quantifiable by the naked eye.      Movie S1. Demonstration video of the operation procedure using the rapid mode. MMPs coated with RBD are first mixed with undiluted serum/plasma for 5 min with handshaking to specifically capture the anti-spike RBD IgG. After rinsing using a magnetic rack, the interfering materials such as other human antibodies can be removed before incubating with PMPs coated with anti-human IgG antibody for another for 5 min with handshaking. Next, the solution is dispensed into the loading chamber, and the device is placed vertically. Subsequently, the solution enters the microchannels in the NOA layer and forms PMP accumulation in the trapping channel visible to the naked eye within 10 min.

Movie S2. Magnetic separation of MMPs and PMPs with the presence of anti-spike IgG.
As PMPs-antibodies-MMPs are formed, less free PMPs can flow and be trapped at a particle dam. Thus, the presence of anti-spike IgG shortens the PMP accumulation length.

Movie S3. Magnetic separation of MMPs and PMPs without the presence of anti-spike
IgG. As MMPs and PMPs are not connected, almost all PMPs are allowed to flow and trapped at a particle dam, causing a longer PMP accumulation length.
Movie S4. PMP accumulation as a monolayer in the trapping channel. The PMPs accumulate into a monolayer in the channel with height of 25 ± 0.3 μm, which maximizes the visibility of PMP accumulation length.

Data processing and analysis
• Linear regression A linear model, y = b0 + b1x, is chosen to perform least-squares regression, where y represents the result of assays, i.e., optical absorbance, trapping length of the PMP accumulation, or OD450 value, and x represents the concentration of SARS-CoV-2 antibody. In the linear regression, the estimates of the intercept , the slope , their variances , and the residual variance of the regression is determined by: where n is the total number of data point calculated by = ∑ , k is the number of concentration levels, is the times of repetition at each concentration level, ̅ and , is the mean value of x and y, as shown below: and ̑ is the predicted value of y for a particular concentration xi, as shown in equation: Hence, the calibration curve is expressed as: where α, is the critical value of student t distribution, which is selected as 1.645 for the 90% confidence interval of two-tailed hypothesis (α = 0.05), and 1/m is the contributes of uncertainty from the average of m replicates in future observation (46, 48).

• Limit of detection
For the estimation of the limits of detection xD, a non-central t-distribution model is selected with the equation: where δ α,β, is the non-centrality value of the non-central t-distribution taking protection against both type I error rate (α, false-positive) and type II error rate (β, false-negative) (46, 48). All LOD was determined based on an appropriate linear range evaluated by the R 2 .

• Accuracy
The measured antibody level by microfluidic chip (Y) is compared with that by the gold standard method ELISA (X) to study the accuracy. The agreement between two sets of measured antibody levels of the 91 volunteers' plasma sample is quantified using Lin's concordance correlation coefficient (ρ̑ ), as shown below (55): where μ and μ are the means, σ and σ are the variances, and ρ is Pearson's correlation coefficient.